Kevin J. Yarema

The effect of nanofiber-guided cell alignment on the preferential differentiation of neural stem cells

Stem cells display sensitivity to substrate presentation of topographical cues via changes in cell morphology. These biomechanical responses may be transmitted to the nucleus through cytoskeletal-linked signaling pathways. Here we investigate the influence of aligned substratum topography on the cell morphology and subsequently, the neuronal differentiation capabilities of adult neural stem cells (ANSCs). ANSCs that were cultured on aligned fibers elongated along the major fiber axis. Upon induction of differentiation with retinoic acid, a higher fraction of cells on aligned fibers exhibited markers of neuronal differentiation as compared with cells on random fiber or unpatterned surfaces. This effect was in part due to substrate selectivity, whereby aligned fiber substrates were less receptive to the attachment and continued survival of oligodendrocytes than random fiber or unpatterned substrates. Substrate-induced elongation alone was also effective in upregulating canonical Wnt signaling in ANSCs, which was further potentiated by retinoic acid treatment. These findings suggest a mechanism by which morphological control of stem cells operates in concert with biochemical cues for cell fate determination.

Metabolic Delivery of Ketone Groups to Sialic Acid Residues APPLICATION TO CELL SURFACE GLYCOFORM ENGINEERING

The development of chemical strategies for decorating cells with defined carbohydrate epitopes would greatly facilitate studies of carbohydrate-mediated cell surface interactions. This report describes a general strategy for engineering the display of chemically defined oligosaccharides on cell surfaces that combines the concepts of metabolic engineering and selective chemical reactivity. Using a recently described method (Mahal, L. K., Yarema, K. J., and Bertozzi, C. R. (1997) Science 276, 1125–1128), we delivered a uniquely reactive ketone group to endogenous cell surface sialic acid residues by treating cells with the ketone-bearing metabolic precursor N-levulinoylmannosamine (ManLev). The ketone undergoes highly selective condensation reactions with complementary nucleophiles such as aminooxy and hydrazide groups. The detailed quantitative parameters of ManLev metabolism in human and nonhuman-derived cell lines were determined to establish a foundation for the modification of cell surfaces with novel epitopes at defined cell-surface densities. Ketones within the glycoconjugates on ManLev-treated cells were then reacted with synthetic aminooxy and hydrazide-functionalized carbohydrates. The remodeled cells were endowed with novel lectin binding profiles as determined by flow cytometry analysis. The simplicity and generality of this method make it well suited for use in the study of carbohydrate-mediated cell surface interactions.

Roles for UDP-GlcNAc 2-Epimerase/ManNAc 6-Kinase outside of Sialic Acid Biosynthesis MODULATION OF SIALYLTRANSFERASE AND BiP EXPRESSION, GM3 AND GD3 BIOSYNTHESIS, PROLIFERATION, AND APOPTOSIS, AND ERK1/2 PHOSPHORYLATION

Roles for UDP-GlcNAc 2-epimerase/ManNAc 6-kinase (GNE) beyond controlling flux into the sialic acid biosynthetic pathway by converting UDP-GlcNAc to N-acetylmannosamine are described in this report. Overexpression of recombinant GNE in human embryonic kidney (HEK AD293) cells led to an increase in mRNA levels for ST3Gal5 (GM3 synthase) and ST8Sia1 (GD3 synthase) as well as the biosynthetic products of these sialyltransferases, the GM3 and GD3 gangliosides. Conversely, down-regulation of GNE by RNA interference methods had the opposite, but consistent, effect of lowering ST3Gal5 and ST8Sia1 mRNAs and reducing GM3 and GD3 levels. Control experiments ensured that GNE-mediated changes in sialyltransferase expression and ganglioside biosynthesis were not the result of altered flux through the sialic acid pathway. Interestingly, exogenous GM3 and GD3 also changed the expression of GNE and led to reduced ST3Gal5 and ST8Sia1 mRNA levels, demonstrating a reciprocating feedback mechanism where gangliosides regulate upstream biosynthetic enzymes. Cellular responses to the GNE-mediated changes in ST3Gal5 and ST8Sia1 expression and GM3 and GD3 levels were investigated next. Conditions that led to reduced ganglioside production (e.g. short hairpin RNA exposure) stimulated proliferation, whereas conditions that resulted in increased ganglioside levels (e.g. recombinant GNE and exogenous gangliosides) led to reduced proliferation with a concomitant increase in apoptosis. Finally, changes to BiP expression and ERK1/2 phosphorylation consistent with apoptosis and proliferation, respectively, were observed. These results provide examples of specific biochemical pathways, other than sialic acid metabolism, that are influenced by GNE.

The homozygous M712T mutation of UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase results in reduced enzyme activities but not in altered overall cellular sialylation in hereditary inclusion body myopathy

Hereditary inclusion body myopathy (HIBM) is a neuromuscular disorder, caused by mutations in UDP‐N‐acetylglucosamine 2‐epimerase/N‐acetylmannosamine kinase, the key enzyme of sialic acid biosynthesis. In Middle Eastern patients a single homozygous mutation occurs, converting methionine‐712 to threonine. Recombinant expression of the mutated enzyme revealed slightly reduced N‐acetylmannosamine kinase activity, in agreement with the localization of the mutation within the kinase domain. B lymphoblastoid cell lines derived from patients expressing the mutated enzyme also display reduced UDP‐N‐acetylglucosamine 2‐epimerase activity. Nevertheless, no reduced cellular sialylation was found in those cells by colorimetric assays and lectin analysis, indicating that HIBM is not directly caused by an altered overall expression of sialic acids.

Gangliogenesis in the enteric nervous system: Roles of the polysialylation of the neural cell adhesion molecule and its regulation by bone morphogenetic protein‐4

The neural crest–derived cells that colonize the fetal bowel become patterned into two ganglionated plexuses. The hypothesis that bone morphogenetic proteins (BMPs) promote ganglionation by regulating neural cell adhesion molecule (NCAM) polysialylation was tested. Transcripts encoding the sialyltransferases, ST8Sia IV (PST) and ST8Sia II (STX), which polysialylate NCAM, were detectable in fetal rat gut by E12 but were downregulated postnatally. PSA‐NCAM‐immunoreactive neuron numbers, but not those of NCAM, were developmentally regulated similarly. Circular smooth muscle was transiently (E16–20) PSA‐NCAM‐immunoreactive when it is traversed by migrating precursors of submucosal neurons. Neurons developing in vitro from crest‐derived cells immunoselected at E12 with antibodies to p75NTR expressed NCAM and PSA‐NCAM. BMP‐4 promoted neuronal NCAM polysialylation and clustering. N‐butanoylmannosamine, which blocks NCAM polysialylation, but not N‐propanoylmannosamine, which does not, interfered with BMP‐4‐induced neuronal clustering. Observations suggest that BMP signaling enhances NCAM polysialylation, which allows precursors to migrate and form ganglionic aggregates during the remodeling of the developing ENS. Developmental Dynamics 236:44–59, 2007.

Characterizing glycosylation pathways

Numerous factors that influence cell-surface carbohydrate composition remain to be elucidated. The combination of novel biochemical and metabolism-based approaches with emerging genomic methods promises to accelerate efforts to understand glycosylation.

Metabolic Flux Increases Glycoprotein Sialylation: Implications for Cell Adhesion and Cancer Metastasis

This study reports a global glycoproteomic analysis of pancreatic cancer cells that describes how flux through the sialic acid biosynthetic pathway selectively modulates a subset of N-glycosylation sites found within cellular proteins. These results provide evidence that sialoglycoprotein patterns are not determined exclusively by the transcription of biosynthetic enzymes or the availability of N-glycan sequons; instead, bulk metabolic flux through the sialic acid pathway has a remarkable ability to increase the abundance of certain sialoglycoproteins while having a minimal impact on others. Specifically, of 82 glycoproteins identified through a mass spectrometry and bioinformatics approach, ∼31% showed no change in sialylation, ∼29% exhibited a modest increase, whereas ∼40% experienced an increase of greater than twofold. Increased sialylation of specific glycoproteins resulted in changes to the adhesive properties of SW1990 pancreatic cancer cells (e.g. increased CD44-mediated adhesion to selectins under physiological flow and enhanced integrin-mediated cell mobility on collagen and fibronectin). These results indicate that cancer cells can become more aggressively malignant by controlling the sialylation of proteins implicated in metastatic transformation via metabolic flux.

Metabolic selection of glycosylation defects in human cells

Changes in glycosylation are often associated with disease progression, but the genetic and metabolic basis of these events is rarely understood in detail at a molecular level. We describe a metabolism-based approach to the selection of mutants in glycoconjugate biosynthesis that provides insight into regulatory mechanisms for oligosaccharide expression and metabolic flux. Unnatural intermediates are used to challenge a specific pathway, and cell surface expression of their metabolic products provides a readout of flux in that pathway and a basis for selecting genetic mutants. The approach was applied to the sialic acid metabolic pathway in human cells, yielding novel mutants with phenotypes related to the inborn metabolic defect sialuria and metastatic tumor cells.

Metabolic labeling of glycoproteins with chemical tags through unnatural sialic acid biosynthesis.

Publisher Summary The importance of protein glycosylation is becoming apparent, and much effort has been directed to the investigation of glycoproteins with the aim of identifying the biological functions of the pendant glycans. Chemical synthesis provides access to oligosaccharides and their analogs for structure-function analyses in vitro . The function of cell surface glycoproteins can depend on the spatial context of their presentation. Thus, biological investigations of isolated glycoprotein molecules and those displayed on a cell surface can show different results. So, the extension of a chemical modification route to cell surface oligosaccharides is a valuable addition to the tools of cell surface biochemistry. The chapter describes the strategy for the metabolic introduction of ketone groups into cell surface sialoglycoconjugates, using the unnatural sialic acid precursor N -levulinoylmannosamine (ManLev). ManLev is converted to N -levulinoyl sialic acid (SiaLev) in cultured cells, permitting the chemical attachment of hydrazide- or aminooxy-derivatized probes to cell surface glycoproteins.

A mathematical model to derive N-glycan structures and cellular enzyme activities from mass spectrometric data

Effective representation and characterization of biosynthetic pathways of glycosylation can be facilitated by mathematical modeling. This paper describes the expansion of a previously developed detailed model for N-linked glycosylation with the further application of the model to analyze MALDI-TOF mass spectra of human N-glycans in terms of underlying cellular enzyme activities. The glycosylation reaction network is automatically generated by the model, based on the reaction specificities of the glycosylation enzymes. The use of a molecular mass cutoff and a network pruning method typically limits the model size to about 10,000 glycan structures. This allows prediction of the complete glycan profile and its abundances for any set of assumed enzyme concentrations and reaction rate parameters. A synthetic mass spectrum from model-calculated glycan profiles is obtained and enzyme concentrations are adjusted to bring the theoretically calculated mass spectrum into agreement with experiment. The result of this process is a complete characterization of a measured glycan mass spectrum containing hundreds of masses in terms of the activities of 19 enzymes. In addition, a complete annotation of the mass spectrum in terms of glycan structure is produced, including the proportions of isomers within each peak. The method was applied to mass spectrometric data of normal human monocytes and monocytic leukemia (THP1) cells to derive glycosyltransferase activity changes underlying the differences in glycan structure between the normal and diseased cells. Model predictions could lead to a better understanding of the changes associated with disease states, identification of disease-associated biomarkers, and bioengineered glycan modifications.